The immediate purpose of the proposed research is to purify and subsequently characterize the acetylcholine receptor (AChR) from mammalian tissue. A partially purified preparation (52,000) has been obtained and studied. Ion exchange chromatography, gel filtration, gel electrophoresis, isoelectric focusing, etc. will all be examined for their feasibility as purification aids. Once purified the receptor will be examined as follows: 1. Determine the types and number of binding sites and dissociation constants of the AChR. Correlate the properties of the isolated receptor with the physiological responses recorded for mammalian muscle and electric organ tissue receptors in situ. 2. Initiate a more detailed characterization of the nature of the purified protein (i.e., glycoprotein, proteolipid, subunit composition, amino acid composition and sequence, "polarity", etc. 3. Use fluorescence and infrared spectroscopy, optical rotary dispersion (ORD) or circular dichroism (CD) to examine the secondary structure of the AChR and to monitor any conformational changes which occur upon binding of cholinergic ligands. 4. Prepare an affinity chromatogrphy adsorbent with the receptor as ligand to determine whether, under varying conditions of elution, other proteins or lipids, if any, may exhibit a specific affinity for the receptor. Examine the ability of the receptor or any proteins or lipids isolated from the receptor-affinity chromatography to induce excitable properties in synthetic lipid bilayer membranes. Reconstitution of an excitable system with the required pure lipid and protein components would be the ultimate goal of these studies. BIBLIOGRAPHIC REFERENCES: Moore, W.M. and Brady, R.N., Purification of a Rat Brain Nicotinic Acetylcholine Receptor Protein. Trans. Am. Soc. Neurochem. 8, 121 (1977). Moore, W.M. and Brady, R.N., Partial Purification of a Nicotinic Acetylcholine Receptor Protein from Rat Brain. Fed. Proc. 36, 760 (1977).